Objective: The aim of this study was to explore the effects of intermittent hypoxia on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods: Primary culturing of rat BMSCs was conducted by adherent cell culturing. BMSCs were identified with immunofluorescence staining. Third-generation BMSCs were randomly divided into the control group and intermittent hypoxia group. The two groups were compared in the formation of mineralized bone nodules after 3 weeks of culture and Real-time PCR was performed for testing the mRNA levels of the bone differentiation markers (ALP, Col-1 and BMP-2) in the two groups at 24 h, 48 h and 72 h, respectively. Results: BMSCs were successfully cultured in this trial. Significantly more mineralized nodules of BMSCs, at different time points, were observed in the intermittent hypoxia group than the control group (P<0.001). Moreover, mRNA levels of ALP, Col-1, and BMP-2 in the intermittent hypoxic group were significantly higher than those in the control group (all P<0.01). Conclusion: Intermittent hypoxia improves osteogenic differentiation of BMSCs.